Chilwell suffered a hamstring injury in Chelsea's 2-1 Champions League win over Dinamo Zagreb in midweek.
Blues manager Graham Potter admitted afterwards that Chilwell's World Cup prospects looked bleak.
The former Leicester City left back's worst fears have now been realised after it was confirmed that he will not be able to play a part in England's trip to Qatar.
Earlier on Saturday, a Chelsea statement read: "Following the injury sustained in our recent game against Dinamo Zagreb, Ben has undergone a scan on his hamstring.
"Results show that Ben has suffered a significant injury and the defender is unfortunately expected to miss the World Cup.
"Ben will now begin a rehabilitation programme with the club's medical department."
Last year, Chilwell suffered a serious knee injury in November and did not return until May.
"Following my ACL injury I worked so hard to make sure I was ready for the World Cup," Chilwell wrote on social media after the news was confirmed.
"It has always been a dream of mine. Unfortunately, this won't be possible following my scan results. I'll do all I can to get back playing for Chelsea ASAP."
England manager Gareth Southgate is set to name his 26-man squad for the World Cup next week and will be sweating on the fitness of some of his full backs.
Reece James is also a doubt for the tournament and Kyle Walker is still working his way back to full fitness.
Chilwell has won 17 caps for England and was part of the Three Lions squad at last summer's European championships, although he did not play a single game in their run up to the final.
N'golo Kante is another Chelsea player who has been ruled out entirely of the World Cup through injury.
